Production of Functional Single-Chain Fv Antibodies in the Cytoplasm of Escherichia coli

Jurado, Paola; Ritz, Daniel; Beckwith, Jon; Lorenzo, Vı́ctor de; Fernández, Luis A.

doi:10.1016/s0022-2836(02)00405-9

Cited by 131 publications

(119 citation statements)

References 49 publications

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“…The variants were functional upon extraction from the reducing environment of the E. coli cytoplasm, consistent with the absence of a disulfide bridge. Functional cytoplasmic production is also an advantage compared with standard antibodies, which do not fold correctly in the strongly reducing environment of the E. coli cytoplasm, although this can be overcome by using a strain lacking glutathione oxidoreductase and thioredoxin reductase (24), or the antibody scaffold used is isolated or developed for this purpose (4).…”

Section: Discussionmentioning

confidence: 99%

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Mouratou

Schaeffer

Guilvout

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

116

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We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/ selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.calorimetry ͉ intrabody ͉ ribosome display ͉ Sac7d ͉ type II secretion system

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Section: Discussionmentioning

confidence: 99%

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Mouratou

Schaeffer

Guilvout

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

116

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“…Rad) as described (24). The anti-E-tag monoclonal antibodyperoxidase conjugate (Amersham Biosciences) was applied for luminescent detection of the PtsN-E-tag fusion proteins with the procedure described (24).…”

Section: Methodsmentioning

confidence: 99%

Growth-dependent Phosphorylation of the PtsN (EIINtr) Protein of Pseudomonas putida

Pflüger

2007

Journal of Biological Chemistry

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The nitrogen-related branch of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) of Pseudomonas putida includes the ptsN gene encoding the EII Ntr (PtsN) enzyme. Although the implication of this protein in a variety of cellular functions has been observed in diverse bacteria, the physiological signals that bring about phosphorylation/dephosphorylation of the PtsN protein are not understood. This work documents the phosphorylation status of the EII Ntr enzyme of P. putida at various growth stages in distinct media. Culture conditions were chosen to include fructose (the uptake of which is controlled by the PTS) or glucose (a non-PTS sugar in P. putida) in minimal medium with casamino acids, ammonia, or nitrate as alternative nitrogen sources. To quantify the relative ratio of PtsN/PtsN ϳ P in live cells, we resorted to the in situ electrophoresis of whole bacteria expressing an E-epitope-tagged EII Ntr followed by the fractionation of the thereby released native proteome in a non-denaturing gel. Although the PtsN species phosphorylated in amino acid His 68 was detected under virtually all growth scenarios, the relative levels of the non-phosphorylated form varied dramatically depending on the growth phase and the nutrients available in the medium. The share of phosphorylated PtsN increased along growth in a fashion apparently independent of any trafficking of sugars. The large variations of non-phosphorylated PtsN in different growth conditions, in contrast to the systematic excess of the phosphorylated PtsN form, suggested that the P-free PtsN is the predominant signaling species of the protein.

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“…Furthermore, the secretory expression of most scFv and Fab fragments involves insoluble aggregated forms called inclusion bodies in the periplasm (14,16), and some antibody fragments cannot be expressed even as inclusion bodies (17). Consequently, a few other systems have been developed for the production of functional antibodies from E. coli, for example, direct production of functional scFv in the cytoplasm by the coexpression of disulfide bond chaperones (18), and refolding of recombinant antibody fragments from inclusion bodies in vitro (19).…”

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confidence: 99%

How Additives Influence the Refolding of Immunoglobulin-folded Proteins in a Stepwise Dialysis System

Umetsu¹,

Tsumoto²,

Hara³

et al. 2003

Journal of Biological Chemistry

161

100

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The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and L-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 M GdnHCl to 1 M GdnHCl, and 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid dipotassium salt binding analysis indicated that the addition of L-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether L-arginine was present. Spontaneous folding at the 1 M GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of Larginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of L-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether L-arginine was present. Controlled coupling of the effects of GSSG and L-arginine led to the complete refolding of scFv in the stepwise dialysis.The internal disulfide linkage in the immunoglobulin fold is of particular importance for most proteins in the immunoglobulin superfamily because this linkage has a critical influence on the stability of these proteins (1, 2). The linkage, which is highly conserved, connects two ␤-sheets in a sandwich structure (3-6). In the case of antibody molecules, Goto and Hamaguchi (1, 7) were the first to analyze intrachain disulfide bond formation in the constant fragments of the immunoglobulin light chain (C L ), 1 demonstrating the contribution of the disulfide linkage to the stabilization and folding of the C L domain. For the variable fragment in antibody molecules (Fv), which is composed of heavy and light chain domains (V H and V L , respectively), the immunoglobulin fold is known to be partially and irreversibly denatured to an aggregate under reducing conditions (2); furthermore, the instability of the Fv that is missing its disulfide linkages has been proved by natural a...

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Production of Functional Single-Chain Fv Antibodies in the Cytoplasm of Escherichia coli

Cited by 131 publications

References 49 publications

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

Growth-dependent Phosphorylation of the PtsN (EIINtr) Protein of Pseudomonas putida

How Additives Influence the Refolding of Immunoglobulin-folded Proteins in a Stepwise Dialysis System

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